This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Spirochetes are a medically significant but a remarkable poorly understood group of bacteria. These organisms cause a variety of important diseases including syphilis, Lyme disease, relapsing fever, and leptospirosis. They are also implicated in periodontal disease and human diarrheal disease. The investigators aim to answer certain questions relating to the structure of spirochete periplasmic flagella (PFs) and motility. Spirochete PFs are the most complex of any bacterial flagellar filament. On the surface of the PFs is a protein sheath comprised of one to two FlaA protein species. The core is comprised of a polymer of three to four protein species referred to as FlaB proteins. The function of the individual PF proteins is not understood. In previous work with the RVBC, isolated PFs from mutant flagella were studied by cryo-EM, and a manuscript is in preparation. In this study, we wish to confirm findings in flagella from intact cells, by comparing various mutant strains having altered motility. These studies are especially relevant in that flagella and/or motility are implicated as a virulence factor for spirochetes and several other species of pathogenic bacteria. In the previous reporting period, four strains of Borrelia burgdorferi were plunge-frozen as whole mounts for cryo-tomography: B31 wild type, Che A, Che X, and Fli G1. Tomographic reconstructions of all four strains were made. Dr. Charon visited the Resource to study the results, and to observe the specimens at the electron microscope in order to help identify the optimal examples for future work. We found the arrangement flagella in native cells to differ from that seen previously in chemically-fixed cells, and the 3-D arrangement of the motile apparatus could be much better appreciated in the tomograms of whole cells, compared to the random thin sections previously studied.